Volume 14 Number 23 1986
Nucleic Acids Research
Adenovirus-2 Ela and Elb gene products regulate enhancer mediated transcription
Venkatachala Natarajan
Laboratory of Biology of Viruses, National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, MD 20892, USA
Received 11 July 1986; Revised and Accepted 4 November 1986
Abstract
We have shown that adenovirus-2 early region 1 gene products in trans and
the SV40 enhancer in cis have an additive effect in stimulating
transcription from adenovirus IVa2 and major late promoters (Natarajan, V.
and Salzman, N. P. , Nucleic Acids Research 13, 4067, 1985). In the present
study, we show that both the Ela and Elb gene products are necessary for
this stimulatory effect on enhancer mediated transcription. In the absence
of Elb region, the transcription is strongly suppressed by Ela.
Transcription from Ela promoter is also stimulated 4-5 fold in the presence
of Elb region. The data suggest that 21K protein coded by the Elb region
modulates transcription from the Ela promoter and the action of Ela gene
products on transcription from other promoters.
INTRODUCTION
The regulation of eukaryotic gene expression has been shown to exist at
many levels, including transcription, RNA processing and raRNA stability (1).
Recent studies have identified a variety of DNA sequences which are
Important for efficient transcription (2-4). In addition, many factors
which control and regulate the transcription have also been characterized
(5-8). Among these are proteins encoded by a number of viral oncogenes
which have the ability to stimulate transcription (6-8). The region of the
adenovirus genome responsible for oncogenic transformation consists of two
transcriptional units, early region la (Ela) and early region lb (Elb) (9).
Studies by many groups have demonstrated that the Ela coded proteins
stimulate transcription from other adenovirus genes and also from a number
of cellular genes (10-18). However, attempts to locate the site of action
of Ela gene products on promoters have not identified a sequence specific
for Ela stimulateion (15, 19, 20). Also, the mechanism by which Ela gene
products stimulate transcription is not fully understood. It has been
proposed that stimulation of transcription can be mediated through the
inactivation of transcriptional repressors (21).
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In addition to its stimulatory activity, the Ela gene products are also
shown to repress enhancer mediated transcription (22-24). This repression
has been observed with polyona, SV40 and adenovirus enhancers and also with
cellular immunoglobulin enhancer (22-24). Interestingly, the Ela gene
products repress the enhancer mediated transcription in certain cells but
not in others (25, 26). For example, the mouse immunoglobulin heavy chain
enhancer mediated transcription is repressed in lymphoid cells but activated
in fibroblast cells (26). Based on this finding, it has been proposed that
certain cells have transcriptional repressors which have the same target
sequence as the Ela gene products (25, 26).
Recently, we have reported that the presence of Ela gene products in
trans and SV40 enhancer in cis have an additive effect in stimulating RNA
synthesis from the adenovirus major late promoter (MLP) and IVa2 promoter
(27). The plasmid pGC212 which was used in our experiments as a source of
Ela gene contains the left end of adenovirus-2 DNA from nucleotides 310 to
2803 and includes the 5'-portion of Elb region (28, 29). (The sequences
from 1600 to 4100 constitute the Elb region). This finding suggests that
Elb gene products were able to modulate regulatory effects of Ela on
transcription.
In this report, we show that the Ela gene products inhibit enhancer
mediated transcription only in the absence of Elb region. Also, in the
presence of the Elb region, transcription from the Ela promoter is
stimulated 4-5-fold demonstrating that Elb region has a role in the
regulation of transcription.
MATERIALS AND METHODS
Restriction enzymes, T4 DNA ligase, alkaline phosphatase and T4 DNA
kinase were purchased either from Boehringer-Mannheira Biochemicals, Bethesda
Research Laboratory or from New England Biolabs.
Cells. HeLa cells were maintained in medium containing 10% fetal calf
serum.
Plasmids. The structures of pSVECATMLP, pGC212, and pSVEla have already
been described. The pSVECATMLP is a recombinant plasmid which contains
adenovirus MLP upstream of a bacterial gene coding for chlorajnphenicol
acetyl transferase (CAT) and the SV40 transcriptional enhancer sequences
(27). The MLP present in this plasmid consists of 260 base pairs of DNA
upstream of and 33 base pairs of DNA downstream of the RNA initiation site
and the SV40 enhancer (72 base pair repeats) is located about 50 base pairs
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upstream of HLP (27). The pGC212 contains the adenovirus-2 DNA from
nucleotide position (np) 310 to 2803, whereas the pSVEla contains the
adenovirus-5 DNA from np 1 to 1834 (23, 29). The construction of plasmids
pVN28, pVN25, pVN25I, pVN18 and pGC212A and their structures are described
in Figure 2.
Transfections and RNA analysis. DNA transfections were carried out using
the calcium phosphate precipitation method (30). Twenty micrograms of DNA
were used to transfect cells in a 100 mm dish. RNA was isolated 48 h after
transfection and analyzed by the Sl-nuclease mapping method as described
earlier (27, 31). The 5'-end labeled DNA probes used in Sl-raapping are
described in figure legends.
RESULTS AND DISCUSSION
In order to understand wtiether the Elb sequences have any effect on
SV40 enhancer mediated transcription from adenovirus major late promoter
(MLP), plasraids with and without this region were used in co-transfection
studies. HeLa cells were transfected with the plasmid pSVECATMLP that
contains the SV40 enhancer (72 bp repeat sequences) and the adenovirus-2 MLP
and a second plasraid that contains varying amounts of Ela and Elb sequences.
Total cellular RNA was isolated 48 hr after transfection and the RNA
synthesized from the MLP was measured (Figure 1). Co-transfection with
plasmid pGC212 (which contains adenovirus-2 DNA sequences from nucleotide
310 to 2803) stimulated the RNA synthesis from MLP by about 50%, confirming
our earlier observation (27). In contrast, co-transfection with plasmid
pSVEla, (which contains adenovirus-5 DNA sequences from nucleotide 1 to
1834) inhibited the transcription by more than 80%, which is in agreement
with results of others (23). The opposite regulatory effects of Ela gene
products that had previously been observed thus were based on differences in
the plasmid that provided the Ela proteins in trans (22, 23, 27).
In order to define the regions involved in stimulation of enhancer
mediated transcription, deletion mutants in the Elb region of pGC212 were
constructed. Their structures are presented in Fig. 2 and results obtained
by co-transfection of these plasmids on levels of enhancer mediated
transcription from MLP are shown in Figure 3a and 3b. To correct for
variations in the efficiency of transfection, all experiments were repeated
several times 1n duplicate with at least two different preparations of
plasmid DNA. The plasraid pVN28, which had the identical adenoviral DNA
sequences as that of pGC212, and the plasmid pVN25 which has adenovirus DNA
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3
4
5
'-^—MLP
Figure 1. Sl-nuciease mapping of RNA synthesized from the adenovirus MLP
in HeLa cells transfected with pSVECATMLPT Twelve micrograns of pSVECATMLP
and 8 pg of either adenovirus Ela gene containing plasmids or pSVOCAT were
co-transfected into HeLa cells by the calcium phosphate procedure (30). Two
different plasmids (pSVEla and pGC212) were used as a source for the Ela
gene. The pSVOCAT does not have any eukaryotic promoter sequence (32).
Total cellular RNA was Isolated and RNA synthesized from MLP was estimated
by Sl-nuclease mapping as described in Materials and Methods. The DNA probe
used had the 5'-end label at the EcoRI site present in the CAT gene of
pSVECATMLP (27). With this probe, a DNA of 326 nucleotides will be
protected
(indicated as MLP) by RNA from MLP. Lane 1 contains the
[32P]-labeled DNA marker, lane 2 had only the labeled DNA probe carried
through the entire procedure, lanes 3, 4, and 5 contained the RNA isolated
from HeLa cells co-transfected with pSVOCAT, pGC212 and pSVEla,
respectively.
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498
498
E1a
E1a
pVN28
pVN25
pVN25I
pVN18
1702
1702
E1bA
Figure 2. Structure of various recombinants with deletions in the Elb
region. The plasmid pGC212 was used as starting material in the
construction of Elb deletion mutants. The adenoviral DNA from nucleotide
position (np) 310 to 2803 present in pGC212 was cloned between the EcoRI and
Hj_ndIII sites of plasmid pUC19 to obtain pVN28 (33). The plasmid pVN25 was
constructed by deleting sequences from np 2501 to 2803. The plasmid pVN25I
is similar to pVN25 but the sequences between 1835 and 2501 are present in
the inverted orientation. The pVN18 has sequences only up to np 1835.
pGC212A, a mutant in the Elb region containing an internal deletion between
np 1767 and 1912 was made by restricting pGC212 with BstEII and SstI,
treating with T4 DNA polymerase and ligating the DNA (34). The direction
and start sites for transcription from Ela and Elb promoters are shown. The
nucleotide sequence numbering of adenovirus DNA is according to GENE BANK.
The various restriction enzymes used and their cleavage sites are also
shown. The pGC212A is cloned in pBR322 and all others are cloned in pUC19.
sequences from 310 to 2501, consistently stimulated transcription from MLP
by 1.5 to 3 fold. In contrast, the plasmid pVN18, which has adeno DNA
sequences from 310 to 1835, inhibited the transcription by more than 90%.
The inhibitory effect observed with pSVEla (Figure 1) and pVN18 (Figure 3)
are comparable. This finding, together with the results seen with pVN25 and
pVN28, show that the Elb region, rather than sequences from 1 to 309
regulate the level of RNA synthesized from MLP. In addition, the plasmid
pGC212A which has internal deletions in the Elb coding region and pVN25I
which has part of Elb region in inverted orientation also inhibited the
transcription (Figure 3). These results demonstrate a role for the Elb
region in regulation of enhancer mediated transcription.
The amounts of Ela specific RNAs synthesized from these plasraids in
co-transfection experiments was also estimated and the results are presented
in Figure 4. The amount of Ela specific RNA synthesized from pVN28 is about
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a
1 2
3 4
5
—MLP-
Figure 3. Regulation of transcription of the adenovirus MLP by Ela
and Elb gene products. The RNA synthesized from MLP in HeLa cells
transfected with pSVECATMLP (12 \ig) and co-transfected with a second plasmid
(8 ug) that contains the Ela gene and with deletions in the Elb region
(described in Figure 2) was quantitated by Sl-nuclease mapping. Isolation
and estimation of RNA from HLP was done as described in Figure 1. The
position of DNA protected from Sl-nuclease digestion is identified as MLP.
The results presented in 3a and 3b are from two separate experiments. The
co-transfected plasmids in 3a are pUC19 (lane 1), pVN25 (lane 2), pVN28
(lane 3), pVN18 (lane 4), and pVN25I (lane 5). In 3b, the co-transfected
plasmids are pUC19 (lane 1) and pGC212A (lane 2). Lane 3 contains [ 3 2 P]
labeled DNA marker.
four fold more than pVN18, clearly showing that the presence of the Elb
region stimulates RNA synthesis from the Ela promoter. Ela specific RNA
synthesized from pVN25 is comparable to that of pVN28, demonstrating that
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2
3
4
5
6
7
8
91 01 1
•1a
Figure 4. Regulation of transcription from the adenovirus Ela
promoter by Elb region. HeLa cells were co-transfected with 8 ug each of
different plasmids which have deletions in the Elb region (described in
Figure 2) along with 12 ug of pSVECATMLP. RNA isolation and Sl-nuclease
mapping
were done as described in Figure 1. The DNA probe used had the
[ 32 P] 5'-end label at np 816 (NarT site) and it extended up to np 310 (EcoRI
site). It was prepared using pVN18 (Figure 2). With this probe, a DNA of
318 nucleotides will be protected (indicated as Ela) from Sl-nuclease
digestion by RNA synthesized from the Ela promoter (35). The Elb deletion
mutants used in co-transfections were pGC212A (lanes 2 and 3), pVN28
(lanes 4 and 5 ) , pVN18 (lanes 6 and 7 ) , pVN25 (lanes 8 and 9) and pVN25I
(lanes 10 and 11). Lane 1 had the [32P]labeled Haelll digested <(>X174 DNA as
marker.
the sequences present up to nucleotide position 2501 are sufficient for
increased levels of Ela RNA synthesis. The Elb region can stimulate
transcription from Ela promoter either by functioning as a downstream
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E1A
E1B
rVN 2016
1836 ADO
13S mRNA »J
32K
PVN PVH
2601 280)
- 22S mRNA
21K
55K
i
AUO
12S mRNA
26K
13S mRNA
21K
m.p. o
1
10
1000
1KO
2000
Sol
11797)
11
12
=1
(W2)
PGC212 A
Figure 5. Diagramatic representation early region Ela and Elb of adenovirus
type-2. The major species of mRNAs coded by this region are indicated by
horizontal arrows and the proteins coded by these mRNAs are shown by striped
bars. The position of initiation and termination (only for 21K protein)
codons and the 3'-end point of various deletion mutants (pVN18 as pVN1835,
pVN25 as pVN2501 and pVN28 as pVN28O3) are shown by the vertical arrows.
The lower part of the figure represents the early region 1 of adenovirus DNA
in base pairs (b.p.) and map positions (m.p.). Also shown are the
initiation and termination codons for the URF10 and URF11 present in the
1-strand and the location of the deletion (from np 1767 to 1912) present in
the Elb region of the plasraid pGC212A. The figure is based on and modified
from Subramanian e_t a K (39).
enhancer or providing trans acting factors. If it is functioning as a
downstream enhancer for the Ela promoter, then it would be expected that
these sequences would also enhance transcription in the inverted orientation
(4, 36). Since the level of Ela RNA synthesized from pVN25I is comparable
to that of pVN18, it seems unlikely that the Elb region stimulates Ela RNA
synthesis by a d s acting mechanism; most likely, it functions by
transactivation. The Elb region present in the plasraid pVN28 can code for
at least three different proteins (Figure 5). There are two open reading
frames (called URF10 and URF11) on the leftward transcribed strand
(1-strand). They can code for polypeptides of 14 and 23K respectively,
whose synthesis as well as the functions in adenovirus infection are not
well characterized (37). The third is the 21K protein, coded by an open
reading frame that extends from an AUG codon at 1711 to a UGA codon at 2236.
This protein and Ela gene products have been shown to have a necessary
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1
2
3
4 5 6
872-
603_ _ _ >
-E1b
310-
^
-E1b
281271-
234-
Figure 6. Sl-nuclease mapping of RNA synthesized from the Elb promoter. An
aliquot of RNA from samples used in Figure 4 were also analyzed for the
presence of RNA from Elb promoter. The DNA probe used had the 5'-end label
at np 2204 and extended to np 1008 (Smal site).
The probe was prepared
using pVN25 (see Figure 2). Lane 1 had [32P]labeled DNA as markers. The
DNA band (indicated as Elb) protected from Sl-digestion by RNA synthesized
in cells transfected with pVN25 and pVN28 (lanes 2 and 3) has the expected
length corresponding to the Elb RNA initiation sites at np 1699 and 1702
(35). The plasmid DNAs used in transfections in lanes 4, 5 and 6 were
pVN18, pVN25I and pGC212A respectively. The RNA from cells transfected with
pGC212A protected a DNA of about 295 nucleotides in length from
Sl-nuclease
digestion. This corresponds to the length of DNA from the [32P]-labeled
5'-end to the mismatch between the DNA probe and the RNA coded by this
plasmid.
function in cell transformation (9). In addition to these three proteins,
pVN28 also can code for the ami no-terminal half of the 55K protein (Figure
5). The 21K protein is the most likely candidate as a transactivator since
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pGC212A has the intact coding sequence for the 14K, 23K and ami no-terminal
half of the 55K proteins but not for the 21K protein (Figure 5 ) , but
inhibits MLP transcription rather than activating it. At present, evidence
for synthesis of the 21K protein in transfected cells is indirect.
Synthesis of RNA from the Elb promoter was determined and the results are
presented in Figure 6. The DNA probe used to estimate the Elb specific RNA
had the 5'-end label near the carboxy terminus of the 21K protein
(nucleotide position 2204) and it is protected from Sl-nuclease digestion by
the RNA isolated from cells transfected with pVN25 and pVN28 (Figure 6,
lanes 2 and 3). This demonstrates that the cells transfected with these
plasmids have stable, translatable RNA which can code for almost full length
21K protein. The cells transfected with pGC212A also have a similar level
of Elb specific RNA and the reduction in the length of the Sl-protected
fragment was that expected, based on the deletion it contained (lane 6).
The cells transfected with pVN18 or pVN25I do not have a detectable level of
Elb specific RNA (lanes 4 and 5 ) , because pVN18 does not contain the Elb
region and in pVN25I, the Elb region 1s present in the inverted orientation.
Ela gene products regulate RNA synthesis from the Ela promoter,
possibly by acting on the Ela enhancer (14, 22). Ela gene products are also
known to repress polyoma and SV40 enhancer mediated transcription by acting
at the level of RNA synthesis (22-24). The results obtained with pVN25 and
pVN28 suggest that in the presence of Elb coded factors (probably 21K
protein), this repression is blocked and RNA synthesis from Ela promoter is
stimulated. This conclusion is also in agreement with an earlier
observation that the amount of Ela specific RNA in cells transformed by the
Ela region alone was always less than in cells transformed by both the Ela
and Elb regions (38). Interestingly, such a mechanism seems to operate not
only on the Ela promotor but also on other co-transfected promoters. While
the Ela gene products alone inhibit SV40 enhancer mediated transcription
from MLP, in the presence of the Elb coded factor, such repression is not
only prevented but transcription is further stimulated. Most likely this is
not due to increased synthesis of Ela gene products in the presence of the
Elb region, because it has been demonstrated that increasing levels of Ela
gene products leads to a progressive decrease in enhancer mediated
transcription (22, 23).
It is known that the 21K protein is needed to maintain the Integrity of
viral and cellular DMAs during adenovirus infection and this function can
play a role in the regulation of transcription (39-44). However, this 1s
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unlikely, since similar amounts of pSVECATMLP ONA were present in cells
co-transfected with either pVN18 or pVN28 (data not shown).
The Ela region and the 5'-half of Elb (sequences present in pVN28) are
sufficient for adenoviral DNA mediated cell transformation (9) and both Ela
and Elb proteins are needed for complete transformation (45, 46). How Ela
and Elb proteins transform the cells is not yet fully understood.
Immunological evidence suggests that the 21K protein is not physically
associated with any of the Ela proteins (47). However, Ela proteins
interact with several cellular proteins and this may be important for their
functions (47). Also, it has been shown that binding of a cellular factor
to an adenovirus promoter is enhanced by Ela proteins (48). The 21K protein
may alter any of these interactions and modify the function of Ela proteins.
Acknowledgements
This work would not have been possible without the advice and criticism
of Dr. N. P. Salzman. I thank E. Ziff and N. Jones for providing the Ela
gene containing plasmids, H. S. Ginsberg for critical reading of the
manuscript and M. B. Vasudevachari for his help in some of the experiements.
The invaluable assistance of J. Carolan in the preparation of the manuscript
is also acknowledged.
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